Changes in chromatin states control gene expression during differentiation of stem cells and defects in intestinal stem cells (ISCs) underlie disorders such as colorectal cancer and inflammatory bowel diseases. In spite of recent advances in identification of ISCs, the role of epigenetic modifications in ISC gene expression, function and intestinal disease remains unknown. EZH2, which is part of the Polycomb repressive complex (PRC) 2, is overexpressed in many colorectal cancers. However, the current knowledge of the functions and mechanisms of most epigenetic enzymes (including PRC2) is derived from in vitro studies in cancer cell lines or ES cells. An in vivo validation and understanding of their working is imperative before the potential of such enzymes as drug targets can be fully realized. PRC2 is thought to repress gene expression in stem cells and work with other histone modifications to control stem cell differentiation. We propose to study the role of the PRC2-mediated histone mark H327me3 in Intestinal stem cell gene expression, function and differentiation. To achieve this, I will combine three experimental approaches: ChIP-seq for the H3K27me3 mark in various intestinal cells (Aim 1); genetic deletion of Eed to eliminate PRC2 activity (Aim 2); and RNA-seq for detailed transcriptome analysis in different intestinal cells with intact and disrupted PRC2 function (Aim 3). To understand the dynamic pattern of H3K27me3 modification during ISC differentiation, we have mapped H3K27me3-marked genes using ChIP assays in ISC and differentiated cells from intestinal villi. In order to understand how the H3K27me3 mark controls gene expression, we will perform detailed transcriptome analysis using RNA-seq in ISC and villus cells. By correlating the differential H3k27me3 mark and differential gene expression between stem and differentiated cells, we will obtain original and detailed information about the role of PRC2 in control of intestinal genes. To evaluate PRC2 requirements in ISC function and intestinal diseases, we have deleted the core PRC2 component gene Eed in the whole intestinal epithelium and also specifically in ISCs, using cell specific knock-out mouse models. Eed-null intestines show severe defects and in-depth analysis of this phenotype, together with gene expression analysis of wild type and Eed-null cells will allow us to identify specific genes, signaling pathways, and regulatory mechanisms involved in ISC maintenance and differentiation. Our studies hold promise to reveal new insights into gene regulation during differentiation and mechanistic details of PRC2-mediated chromatin modifications.